The long-term goal of this project is to understand the molecular events by which oxidant stress alters endothelial regulation of vascular reactivity. Vascular endothelial cell (VEC) cytosolic Ca2+ is a key second messenger which contributes to the secretion of vasoactive paracrine substances. This proposal focuses on the effect of oxidant stress on Ca2+ signaling in VECs. The model oxidant is ter-butyl-hydroperoxide (t-bu-OOH). Bradykinin will be employed as the model agonist. t-bu-OOH inhibits Ca2+ signaling in fura-2-loaded VECs (Elliott and Schilling, 1990). t-bu-OOH initially inhibits bradykinin-stimulated Ca2+ influx, with no effect on basal cytosolic Ca2+. After longer incubations with the oxidant, agonist-stimulated release of Ca2+ from internal stores is decreased. t-bu-OOH produces progressive loss of membrane potential, measured by giga- seal, and net loss of cellular K+ and net gain of cellular Na+, determined using radioisotopes. This proposal explores the molecular mechanisms responsible for the above findings. Specific Aim I will characterize the mechanism by which oxidant stress inhibits agonist-stimulated changes in cytosolic Ca2+. The effects of oxidant stress on 1) inositol polyphosphate (IP) production, and 2) Ca2+ release from internal stores by IP3 and/or GTP will be investigated using radiolabeling techniques in intact and saponin-permeabilized VECs. The effect of oxidant stress on the kinetics of ATP-dependent Ca2+ pumps will also be characterized. Specific Aim II will characterize the effect of oxidant stress on ion concentration gradients and ion conductance across the cell membrane. The hypotheses that oxidant stress: 1) stimulates Na+ influx via a non- selective cation channel; 2) stimulates K+ efflux via Ca2+-dependent K+ channels; and 3) inhibits Na+/K+-ATPase, will be investigated using radioisotopic tracers and direct, giga-seal measurements. The link between oxidant inhibition of agonist-stimulated Ca2+ influx and oxidant-induced membrane depolarization will be investigated in VECs dually loaded with the Ca2+-sensitive fluorescent indicator, fura-2, and the potential-sensitive dye, di-4-ANEPPS. Thus, these experiments will characterize how oxidant stress alters endothelial cell Ca2+ signaling. This work will form the basis for the long-term objective which is to understand how oxidant stress alters the regulation of vasoreactivity by VECs.